A Study of mitosis, meiosis, histology, and scanning electron microscopic details of spermatogenesis in an infertile male with probable 46,XY/47,XXY germinal mosaicism

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<ul><li><p>American Journal of Medical Genetics 10:119-131 (1981) </p><p>A Study of Mitosis, Meiosis, Histology, and Scanning Electron Microscopic Details of Spermatogenesis in an Infertile Male With Probable 46,XY/47,XXY Germinal Mosaicism Barathur R. Rajendra, Mingliang Lee, Louis Amorosa, and Leonard J. Sciorra </p><p>Division of Medical Genetics, Department of Pediatrics (B. R. R., M-I. L., L. J.S.), and Department of Medicine (L.A.), Rutgers Medical School, Piscata way, New Jersey </p><p>A 29-year-old infertile man with mild eunuchoid body proportions and unilateral gynecomastia with a low sperm count had normal chromosomes in lymphocytes and skin fibroblasts. Serum testosterone, urinary ketosteroid, and follicle-stimulating hormone levels were normal, but serum prolactin, luteinizing hormone, and estrogen levels were above normal. Testicular biopsy showed hypocellularity, a pacuity of maturing sperm, and increased lipid content in early spermatogonia. Cytogenetic analysis of the testicular material showed probable 46 ,XY /47 ,XXY mosaicism in spermatogonial cells. None of the cells in diakinesis and metaphase I showed the extra- X as a univalent, and 50% of the cells in diakinesis were hypodiploid with intact X Y bivalents. Further analysis of spermatogenesis revealed poly- morphic dyads, triads, tetrads, and uncleaved meiotic products of first and second meiosis. Sex chromatin studies of testicular cells showed that 20% of the cells were X-chromatin positive. Scanning electron micro- scopic study of sperm revealed a wide range of polymorphisms, with some uncleaved meiotic products maturing into double-headed and double- tailed sperm or sperm with bulbous middle pieces or protrusions. A cyto- genetic basis for the origin of these abnormalities is presented. </p><p>Key words: meiosis, spermatogenesis, scanning electron microscopy of sperm, sperm polymorphisms, infertility, germinal mosaicism, Klinefelter syndrome </p><p>INTRODUCTION Between 5% and 15% of all infertile men seen in infertility clinics have a chromo- </p><p>some abnormality [Koulischer and Schoysrnan, 19741 and asperrnia, azoospermia, oligo- </p><p>Received for publication November 27, 1979; revision accepted April 20, 1981. </p><p>Address reprint requests to Ming-liang Lee, MD, PhD, Division of Medical Genetics, Department of Pediatrics, CMDNJ-Rutgers Medical School, Piscataway, NJ 08854. </p><p>0148-7299/81/1002-0119 $04.00 0 1981 Alan R. Liss, Inc. </p></li><li><p>120 Rajendra et a1 </p><p>spermia, asthenospermia, and/or teratospermia due t o abnormal spermatogenesis. One of the most common of these conditions is the Klinefelter syndrome. The manifestations of this syndrome may be reduced by XY mosaicism in somatic tissues. Some mosaic patients have been reported t o produce some motile spermatozoa and claim t o have fathered children [Foss and Lewis, 1971, Neuhauser and Opitz, 19751. </p><p>somatic cells and a probable 46,XY/47,XXY mosaicism in germ cells. We report a eunuchoid, infertile man with a 46,XY chromosome constitution in </p><p>REPORT OF PROPOSITUS </p><p>The propositus is a 29-year-old infertile man with normal physical and mental development and an unremarkable medical history. The patient has normal erections, ejaculations, and libido. He shaves daily. His sperm count was 1.2-2.0 million/cc; motility was reduced. The patient was treated with two injections of Depotestosterone (400 mg) 3 4 days apart; two months later sperm counts were not improved. The patients wife is normal and conceived after artificial insemination by donor. </p><p>Physically, the patient was healthy, well developed and of normal intelligence. Height was 183 cm and span was 187 cm; UBSlLBS ratio was 88/95 (0.92). (Fathers height is 183 cm and brothers height is 185 cm.) Facial, axillary, and pubic hair distribu- tion was normal; however, little hair was present on the chest or upper limbs. (Patients father and brother are very hairy.) The skin was soft, and the amount of subcutaneous fat was greater than normal. There was fullness of the left nipple, with palpable glandular tissue, but n o galactorrhea. External genitalia appeared normal. Both testes measured 19.7 cm3 (normal range 10.7-21.1 cm3) and were firm; the prostate was firm, smooth, and of normal size. </p><p>Endocrine studies showed the following results: serum testosterone level 540 ng/dl (normal 300-1,200); serum prolactin 41.6 ng/ml (7-1 8) ; urinary LH 35 IU/day (5-20); urinary FSH 1 5 IU/day (2-15); ketosteroids 14.4 mg/day (10-23); and total estrogens 136 mcg/day (4-25). </p><p>MATERIALS AND METHODS </p><p>Chromosome Analysis Routine chromosome analysis on cultured lymphocytes (banded and unbanded) and </p><p>buccal smear analysis from right and left cheeks was done twice, 11 months apart; 100 metaphases were counted each time, and five karyotypes were prepared from each sample. A skin biopsy was obtained from the left arm, and fibroblast cultures were initiated in routine manner; 1 0 0 fibroblast metaphases were counted, and five karyotypes were pre- pared and analyzed. Lymphocyte and fibroblast chromosomes were also studied by fluorescent methods [Uchida and Lin, 19741. </p><p>Two sections of a testicular biopsy were obtained from the right testis for histo- logical, cytogenetic, and transmission electron microscopy (TEM) studies. For cytogenetic studies, fresh testicular tissue was minced with a fine scalpel and scissors, incubated in 0.6% sodium citrate solution at 22C for 25 minutes, and processed for chromosome study by the method of Sasaki and Makino [1965] . Slides were also processed for G- and Q-banding. All spermatogonial, diakinesis, and metaphase spreads were counted, photographed, and karyotyped when possible. </p></li><li><p>Meiosis in 46,XY/47,XXY Germinal Mosaicism 121 </p><p>Histological Study In addition to the above studies, very thin sections of the biopsy were processed </p><p>for X-chromatin staining (Feulgen, hematoxylin and eosin) by the methods of Plunkett and Barr [1956] and Myers 119591 ; 300 cells from the tubules were scored for X- chromatin bodies. Testicular biopsy slides from two age-matched normal men (29-3 1 years of age) were used as controls in all comparisons of histology and sex chromatin analysis. </p><p>Scanning Electron Microscopy (SEM) of Sperm After liquefaction at 37C for two hours, the semen samples, obtained after three- </p><p>day abstensions from sex, were washed gently in three changes of Krebs-Ringer phosphate buffer (pH 7.2) at room temperature, with intermittent and mild centrifugation [Could, 19731. The samples were then fixed in 2% glutaraldehyde in 0.05 M phosphate buffer (pH 7.2) and mounted on coverslips treated with poly-L-lysine (PLL), which is a modifica- tion of the method used by Mazia et a1 [1975]. A 0.1% solution of PLL (P-1886, Sigma) in water was dispersed onto coverslips and allowed to stand for two hours in a moist chamber at room temperature. After thorough rinsing in running distilled water, the cover- slips were mounted onto SEM stubs with conductive silver paint with the PLL side face- up. Small drops of the fixed sperm were placed on each of the stubs and left in a moist chamber for two hours. The samples were then dehydrated in gradual increases of ethyl alcohol and critical-point dried. The stubs were then coated with gold-palladium (80 A) and studied at magnifications of 1,000 -1 0,000 in an ETEC Autoscan SEM. One hundred sperm were studied per stub and classified for various abnormalities; 25 sperm were measured for head length and width. Representative pictures were taken. A control sample of semen from a fertile normal donor was processed similarly for SEM studies. </p><p>RESULTS AND DISCUSSION </p><p>Cytogenetic Studies Chromosome analysis of the patients cultured lymphocytes and skin fibroblasts </p><p>showed a 46,XY constitution, without mosaicism or structural abnormalities. No X- chromatin bodies were seen in buccal smear cells. </p><p>Cytogenetic analysis of the testicular biopsy is presented in Table I. A total of 55 spreads were anlayzed. The majority(35) were spermatogonia, and 20 cells were in </p><p>TABLE I. Cytogenetic Data From Testicular Biopsy </p><p>Phase Total 46,XY 47,XXY Hypodiploid Hyperdiploid a) Spermatogonia 35 8 23 2 2 </p><p>Total 22 Bivalents + XY 21 Bivalents + XY Random loss </p><p>b) Diakinesis or metaphase-I 20 10 Cia 4b </p><p>aFour cells had a missing D bivalent; one cell showed a missing B group bivalent; and one cell showed a missing C group bivalent. bThese cells had intact XY bivalents, but the missing bivalents could not be identified. </p></li><li><p>122 Rajendra et a1 </p><p>diakinesis or early metaphase I. Only two second-division metaphase spreads were identi- fied; counts were unclear. Of the 35 spermatogonial spreads, 23 (66%) (Fig. la) had a count of 47 chromosomes, eight had a normal 46,XY count, and four were hypodiploid or hyperdiploid. The extra chromosome was a C-group chromosome. Attempts to G-band these spermatogonial metaphases failed to identify the extra C-group chromosome, but Q-banding identified the Y chromosome. In all 23 spreads with 47 chromosomes, the extra chromosome was of the same C-group size. </p><p>Fig. 1. (a) Spermatogonial spread with karyotype showing 47 chromosomes. (b) Spennatogonial spread with karyotype showing 46 chromosomes. </p></li><li><p>Meiosis in 46,XY/47,XXY Germinal Mosaicism 123 </p><p>Of the 20 cells in diakinesis or metaphase I (Table Ib), ten were normal with 22 bivalents + XY (Fig. 2a), six showed 21 bivalents + XY constitution, and among these four cells showed missing D-group bivalents (Fig. 2b), one cell had a missing C-group bivalent, and one cell had a missing B-group bivalent. The four remaining cells showed random loss of autosomal bivalents with intact XY bivalents. None of these cells showed the extra C-group chromosome. </p><p>Fig. 2. (a) Cell in diakinesis with 22 I1 + XY. (b) Cell in diakinesis with 21 I1 + XY. </p></li><li><p>124 Rajendra et a1 </p><p>Histological Studies of Testicular Biopsy </p><p>tubules. Other areas showed relatively normal-appearing tubules with reduced number of mature sperm and increased lipid content in spermatogonia. There was no interstitial fibrosis or apparent Leydig cell hyperplasia. Sex chromatin analysis was done on very thin sections of the biopsy using Feulgen or cresyl violet staining. Hematoxylin and eosin stained and toluidine stained slides served as controls. Of 300 Sertoli, Leydig, and germ cell interphase nuclei analyzed, 59 were X-chromatin positive (Fig. 3 a-d). X-chro- matin studies of testicular biopsies from age-matched controls (from established normal fertile males) did not show X-chromatin bodies in Leydig, Sertoli, or germ cell interphase nuclei. </p><p>These X-chromatin studies, and the patients phenotype and endocrine abnor- malities, suggest that the extra C chromosome seen in 66% of the spermatogonial spreads was an X chromosome and that the patient had 46,XY/47,XXY germinal mosaicism. The fact that we did not detect the extra X chromosome in any of the diakinesis or metaphase I spreads either as univalent, trivalent, or fragment(s) in- dicates that either 1) the X chromosome is lost, 2) such cells do not go through meiotic division, or 3) we failed to see such cells in our slides. However, Neuhauser and Opitz [1975] report that spermatogonia with 47,XXY can go through meiotic division. Their patient was a man with 46,XY/47,XXY somatic mosaicism with an atrophic testis and a 47,XXY son. A similar discrepancy was observed between sperma- togonial counts and diakinesis or metaphase I counts in several 47,XYY men. The expected extra Y chromosome was never identified in diakinesis or metaphase I spreads by Melnyk et a1 [1969], Evans et a1 [1970], or Hulttn [1970]. In one case, Hulttn [ 19701 reported X Y / X W mosaicism in spermatogonial cells, which was judged on the basis of six G-sized chromosomes in some spermatogonial metaphases with 47 chromo- somes. These results are very similar to our studies with a 46,XY/47,XXY mosaicism in germinal cells. Since the spermatogonial cells showed mosaicism, it is possible that this in itself causes meiotic instability (Table Ib). </p><p>In our patients testicular biopsy, we found a number of first-division and second-division chromatin and cell size polymorphisms, which included dyads, triads, and tetrads (Fig. 4 a-d). In plants, cell and gamete size polymorphisms arising from meiotic irregularities due to aneuploidy, pairing problems in prophase I , and disruption of meiotic spindles may result in sterility [Dover, 1972; Rajendra et al, 19781. Thus, the origin of cell size polymorphisms in our patient could be due to 47,XXY sperma- togonial cells or aneuploid diakinesis. It is reasonable to assume that aneuploid germ cells give rise to gamete polymorphisms. These polymorphisms should be reflected at maturity as sperm size polymorphisms. Such findings were indeed apparent when the sperm were analyzed via SEM (see below). </p><p>studies. Furthermore, evidence of polymorphic differentiation was noted in the TEM studies (Fig. 5) and was confirmed in the SEM studies of sperm. </p><p>Light microsocpy showed areas of atrophy and hyalinization of the seminiferous </p><p>Material submitted for TEM analysis showed findings consistent with light microscopic </p><p>Light Microscopic and Scanning Electron Microscopic Studies of Sperm </p><p>results. Sperm motility varied between 35% and 50%. At least three different sperm sizes were observed. </p><p>Light microscopic studies of sperm on three different occasions yielded different </p></li><li><p>Meiosis in 46,XY/47,XXY Germinal Mosaicism 125 </p><p>Fig. 3. (a) Two Leydig cells, one of which is sex chromatin positive (Feulgen staining; magnifica- tion, X 1,250). (b) A sex chromatin positive cell that is probably a secondary spermatocyte (Feulgen staining; magnification, X 1,250). (c) Sex chromatin positive and negative Sertoli cells. (Feulgen staining; magnification, X 1,250). (d) Cresyl violet-stained Leydig cells, one of which is sex chromatin positive (magnification, X 1,250). </p><p>Fig. 4. (a) A polymorphic dyad at the end of meiosis I. Note the area of noncleavage and distribu- tion of chromatin. (b) A polymorphic triad at the end of meiosis 11. Note the point of noncleavage and micro-nuclei. (c) Half of a tetrad at the end of meiosis I1 showing noncleavage. (d) Half of a tetrad at the end of meiosis I1 showing differences in size of gametes (polymorphic). </p></li><li><p>126 Rajendra et a1 </p><p>Fig. 5. A transmission electron microscopic picture of a section of testicular biopsy from patient showing abnormal sperm differentiation (magnification, X 8,000). </p><p>TABLE 11. Measurements of Sperm Heads (Width and Length) From the Patient and Normal Control Sperm* </p><p>Width Gum) Range Length Gum) Range </p><p>Control </p><p>Patient </p><p>~ ~~ </p><p>2.4 k 0.023a (2.2-2.6) 3.4 f 0.025 (3.0-3.6) 2.7 k 0.098 (2.0- 3.2) 3.6 * 0.121 (2.3-4.5) </p><p>*Twenty-five sperm from controls and 25 sperm from the patient were randomly selected, photographed, and measured. aStandard error. </p><p>Various kinds of sperm cell size polymorphisms were detected in the SEM studies. Compared to control sperm from a normal fertile donor, our patients sperm showed higher head size variation (Table 11, Figs. 6 and 7). The length of the...</p></li></ul>